Reduction of anthranilic acid to 2-aminobenzaldehyde by the white-rot fungus Bjerkandera adusta DSMZ 4708.

The biocatalytic aerobic "in-water" reduction of anthranilic acid to 2-aminobenzaldehyde by growing cultures of the basidiomycetous white-rot fungus Bjerkandera adusta has been studied. The high specific activity of Bjerkandera adusta towards the carboxylic group of anthranilic acid that allows avoiding the formation of the corresponding alcohol has been demonstrated using different substrate concentrations. The presence of ethanol as co-solvent allows increasing the yield of target product. In contrast to chemical reducing agents that usually yield 2-aminobenzyl alcohol, an overreduction of anthranilic acid is completely suppressed by the fungus and gives the target flavor compound in satisfactory preparative yields. It was shown that the activity of Bjerkandera adusta towards anthranilic acid does not apply to its m- and p-isomers.

Enhanced Efficiency of the Removal of Cytostatic Anthracycline Drugs Using Immobilized Mycelium of Bjerkandera adusta CCBAS 930.

The aim of this study was to evaluate the bioremoval of anthracycline antibiotics (daunomycin-DNR, doxorubicin-DOX, and mitoxantrone-MTX) by immobilized mycelium of B. adusta CCBAS 930. The activity of oxidoreductases: versatile peroxidases (VP), superoxide dismutase (SOD), catalase (CAT), and glucose oxidase (GOX), and the levels of phenolic compounds (PhC) and free radicals (SOR) were determined during the biotransformation of anthracyclines by B. adusta strain CCBAS 930. Moreover, the phytotoxicity (Lepidium sativum L.), biotoxicity (MARA assay), and genotoxicity of anthracyclines were evaluated after biological treatment. After 120 h, more than 90% of anthracyclines were removed by the immobilized mycelium of B. adusta CCBAS 930. The effective biotransformation of anthracyclines was correlated with detoxification and reduced genotoxicity.

Removal of Aflatoxin B1 by Edible Mushroom-Forming Fungi and Its Mechanism.

Aflatoxins (AFs) are biologically active toxic metabolites, which are produced by certain toxigenic Aspergillus sp. on agricultural crops. In this study, five edible mushroom-forming fungi were analyzed using high-performance liquid chromatography fluorescence detector (HPLC-FLD) for their ability to remove aflatoxin B1 (AFB1), one of the most potent naturally occurring carcinogens known. Bjerkandera adusta and Auricularia auricular-judae showed the most significant AFB1 removal activities (96.3% and 100%, respectively) among five strains after 14-day incubation. The cell lysate from B. adusta exhibited higher AFB1 removal activity (35%) than the cell-free supernatant (13%) after 1-day incubation and the highest removal activity (80%) after 5-day incubation at 40 °C. In addition, AFB1 analyses using whole cells, cell lysates, and cell debris from B. adusta showed that cell debris had the highest AFB1 removal activity at 5th day (95%). Moreover, exopolysaccharides from B. adusta showed an increasing trend (24-48%) similar to whole cells and cell lysates after 5- day incubation. Our results strongly suggest that AFB1 removal activity by whole cells was mainly due to AFB1 binding onto cell debris during early incubation and partly due to binding onto cell lysates along with exopolysaccharides after saturation of AFB1 binding process onto cell wall components.

Chemical Composition and Ultraviolet Absorption Activity of an Aqueous Alkali Extract from the Fruiting Bodies of the Tinder Conk Mushroom, Fomes fomentarius (Agaricomycetes).

White rot mushroom Fomes fomentarius is a medicinal fungus with great potential to be explored. This work focused on the chemical composition of a basic aqueous extract from F. fomentarius fruiting bodies. The extract was mostly composed of phenolics, carbohydrates, minerals, and crude fat with a low amount of proteins and chitin. One-third of the total carbohydrates were in the form of beta-glucans with minor amounts of alpha-glucans. The most valuable essential part of the extract was composed of an acid-resistant ultraviolet (UV)-absorbing mixture of phenolic compounds such as melanins, lignins, and humic acids. These compounds, also referred to as melanin-like pigments, provided for the high antioxidant activity of the extract measured in vitro. Moderate sun-protective capacity was observed with regard to UVB rays and also expected in the UVA range. Quantification of melanin-like pigments in the F. fomentarius extract was possible either gravimetrically as acid-insoluble residue or spectrophotometrically in the UV region. Melanin estimation, based on nitrogen measurements, offered misleading results due to the presence of nitrogen-free melanins along with other nitrogen-containing compounds such as proteins and chitin. F. fomentarius water-soluble basic extract, containing beta-glucans and rich in melanin-like substances, could be used, for example, for topical skin application to prevent cell damage caused by excessive UV exposure or cytotoxic free radicals. The bioactive potential, safety, and further applications of the F. fomentarius extract are currently being investigated.

Optimization of fermentation of Fomes fomentarius extracellular polysaccharide and antioxidation of derivatized polysaccharides.

Fermentation is a metabolic process that converts sugars into acids, gases, or alcohol. This process occurs in yeasts and bacteria, as well as in muscle cells when faced with a lack of oxygen. In this paper, isolation, culture, purification and extracellular polysaccharides of strain Fomes fomentarius were studied. Extraction of polysaccharides from a culture based on F. fomentarius extracellular polysaccharides, extracellular polysaccharides fermentation experiments was optimized and compared, the optimal fermentation method was obtained; extracellular polysaccharides were sulfated, phosphorylated experiments, selenium acidified, discussed the preparation of derivative polysaccharides and microscopic detection, and finally studied extracellular polysaccharides on DPPH, The scavenging ability, superoxide anion radical and hydroxyl radical scavenging ability of the derived polysaccharides were compared. The results showed that the extracellular polysaccharide and derivatized polysaccharide of F. fomentarius had certain antioxidant activity.

Insights into the mechanism of cyanobacteria removal by the algicidal fungi Bjerkandera adusta and Trametes versicolor.

Fungal mycelia can eliminate almost all cocultured cyanobacterial cells within a short time. However, molecular mechanisms of algicidal fungi are poorly understood. In this study, a time-course transcriptomic analysis of algicidal fungus Bjerkandera adusta T1 was applied to investigate gene expression and regulation. A total of 132, 300, 422, and 823 differentially expressed genes (DEGs) were identified at 6, 12, 24, and 48 hr, respectively. Most DEGs exhibited high endopeptidase activity, cellulose catabolic process, and transmembrane transporter activity by using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Many decomposition genes encoding endopeptidases were induced a little later in B. adusta T1 when compared with previously investigated algicidal fungus Trametes versicolor F21a. Besides, the accumulated expression of Polysaccharide lyases8 (PL8) gene with peptidoglycan and alginate decomposition abilities was greatly delayed in B. adusta T1 relative to T. versicolor F21a. It was implied that endopeptidases and enzymes of PL8 might be responsible for the strong algicidal ability of B. adusta T1 as well as T. versicolor F21a.

Immobilization interaction between xenobiotic and Bjerkandera adusta for the biodegradation of atrazine.

The aim of the present work is to evaluate the ability of 'fungi' for the biodegradation of recalcitrant xenobiotic compound, 'Atrazine' in batch liquid cultures. Different parameters like pH (2.0-8.0) temperature (16-32 °C), biomass (1-5 g), and concentration (25-100 ppm) were optimized for the efficient degradation of atrazine. The decomposition behavior of atrazine is analyzed with the help of Fourier Transform Infrared (FTIR) spectroscopy. Herein, we have reported that the Bjerkandera adusta possess high removal efficiency of the xenobiotic compound (atrazine) up to 92%. The fungal strain investigated could prove to be a valuable active pesticide degrading micro-organism, with high detoxification values. These results are useful for improved understanding and prediction of the behavior and fate of B. adusta in the bio-purification of wastewater contaminated with xenobiotics. Thus providing a new and green approach for the remediation of toxicants without altering the environmental components.

Solid state fermentation by Fomitopsis pinicola improves physicochemical and functional properties of wheat bran and the bran-containing products.

Wheat bran was solid state fermented by Fomitopsis pinicola. The results showed that the processing properties were increased by fermentation and the content of total phenol and alkylresorcinols was 5.91 and 1.55 times of the unfermented bran respectively by the 6th day. The total antioxidant capacity was 5.73 times of the unfermented sample by the 4th day. Electronic nose analysis showed that the fermented wheat bran had a special flavor. GC-MS analysis found that 4-ethyl-2-methoxy-phenol was the main flavor substance, which was sharply increased during the fermentation. Furthermore, the textural properties of the dough and bread containing fermented bran were significantly improved. The content of phytic acid in the bread was significantly decreased, while the protein, total phenol and alkylresorcinols contents were significantly increased. Results suggest that solid state fermentation by Fomitopsis pinicola is a promising way to improve wheat bran to a nutritious and flavorful cereal food ingredient.

Fomitopsis officinalis: a Species of Arboreal Mushroom with Promising Biological and Medicinal Properties.

Medicinal mushrooms of the order Polyporales have a long history of use, which is evidenced by the finding of dissected fruiting bodies with Ötzi, who lived over 5000 years ago. Because of its valuable biological properties and its use in 18th and 19th-century pharmacy, Fomitopsis officinalis used to be mass-collected. Moreover, the large demand for larch wood and non-wood materials (resin) caused an excessive exploitation of larch forests, which directly contributed to the disappearance of F. officinalis from its natural environment. The qualities of medicinal preparations obtained from the F. officinalis fruiting bodies are determined by the unique composition of its bioactive compounds, such as: triterpenoids, polysaccharides, organic acids, coumarins and phenolic compounds. It has been proved that both crude extracts and the compounds isolated from F. officinalis have a wide spectrum of therapeutic effects, including anti-inflammatory, cytotoxic, and antimicrobial effects.

Biological Evaluation and In Silico Study of Benzoic Acid Derivatives from Bjerkandera adusta Targeting Proteostasis Network Modules.

Abstract: A main cellular functional module that becomes dysfunctional during aging is the proteostasis network. In the present study, we show that benzoic acid derivatives isolated from Bjerkandera adusta promote the activity of the two main protein degradation systems, namely the ubiquitin-proteasome (UPP) and especially the autophagy-lysosome pathway (ALP) in human foreskin fibroblasts. Our findings were further supported by in silico studies, where all compounds were found to be putative binders of both cathepsins B and L. Among them, compound 3 (3-chloro-4-methoxybenzoic acid) showed the most potent interaction with both enzymes, which justifies the strong activation of cathepsins B and L (467.3 ± 3.9%) on cell-based assays. Considering that the activity of both the UPP and ALP pathways decreases with aging, our results suggest that the hydroxybenzoic acid scaffold could be considered as a promising candidate for the development of novel modulators of the proteostasis network, and likely of anti-aging agents.

Formation of Decatrienones with a Pineapple-like Aroma from 1-13C-Acetate by Cell Cultures of the Birch Polypore, Fomitopsis betulina.

During submerged cultivation, the edible basidiomycete Fomitopsis betulina (previously Piptoporus betulinus) developed a fruity odor, strongly reminding of pineapple. Olfactometric analysis showed that this impression was mainly caused by the two (5E/Z,7E,9)-decatrien-2-ones. At the time of maximum concentration on the 5th day, the (5E/5Z)-ratio was 94:6. Three hypotheses were experimentally examined to shed light onto the genesis of the uncommon volatiles: first, an indirect effect of agro-industrial side-streams, such as cabbage cuttings, supporting good growth; second, an unsaturated odd-numbered fatty acid precursor; and third, a polyketide-like pathway. In the presence of 1-13C- or 2-13C-acetate up to five acetates were incorporated into the molecular ions of the C10-body. Addition of 1-13C-pyruvate or 1-13C-lactate did not confirm an odd-numbered starter of the polyketide chain. None of the methylketones was found in pineapple or any other food before.

Biotransformation and toxicity effect of monoanthraquinone dyes during Bjerkandera adusta CCBAS 930 cultures.

The aim of this study was to evaluate of possibility of biotransformation and toxicity effect of monoanthraquinone dyes in cultures of Bjerkandera adusta CCBAS 930. Phenolic compounds, free radicals, phytotoxicity (Lepidium sativum L.), ecotoxicity (Vibrio fischeri) and cytotoxicity effect were evaluated to determine the toxicity of anthraquinone dyes before and after the treatment with B. adusta CCBAS 930. More than 80% of ABBB and AB129 was removed by biodegradation (decolorization) and biosorption, but biodegradation using oxidoreductases was the main dye removing mechanism. Secondary products toxic to plants and bacteria were formed in B. adusta strain CCBAS 930 cultures, despite efficient decolorization. ABBB and AB129 metabolites increased reactive oxygen species (ROS) production in human fibroblasts, but did not increase LDH release, did not affect the resazurine reduction assay and did not change caspase-9 or caspase-3 activity.

Cultivation and Medicinal Value of the Red Belt Conk Mushroom Fomitopsis pinicola (Agaricomycetes).

This study examined biological characteristics, liquid fermentation, and cultivation of Fomitopsis pinicola. A single-factor test concluded that the optimal carbon and nitrogen sources for mycelial growth were soluble starch and yeast paste; the optimal culture temperature was 31°C, and the optimal pH was 6.0. The orthogonal experiment indicated that the optimal formula for mycelial culture was 25 g soluble starch, 2 g yeast extract, 1 g KH2PO4, and 1.5 g MgSO4 added to 1 L water. The optimal conditions for liquid fermentation culture consisted of the following: a loading volume 90 mL, inoculation volume 30 mL, and rotation speed 160 rpm. The optimal substrate formula for domestic culture was 20% corn cob, 30% sawdust, 20% wheat bran, 25% cotton seed shell, 3% corn meal, 1% gypsum, and 1% lime, which produced the highest yield of fruiting bodies. The results provided basic data for deep liquid fermentation culture and recommendations for the further development and utilization of F. pinicola.

Fungal biodegradation and multi-level toxicity assessment of vinasse from distillation of winemaking by-products.

The wastewaters from distilleries of winemaking by-products, a scarcely studied type of vinasse, were treated by white-rot fungal strains from species Irpex lacteus, Ganoderma resinaceum, Trametes versicolor, Phlebia rufa and Bjerkandera adusta. The main objectives of this study were to evaluate fungal performance during vinasse biodegradation, their enzyme patterns and ecotoxicity evolution throughout treatment. Despite all strains were able to promote strong (>80%) dephenolization and reduction of total organic carbon (TOC), P. rufa was less affected by vinasse toxicity and exhibit better decolorization. In batch cultures at 28 °C and pH 4.0, the first phase of P. rufa biodegradation kinetics was characterized by strong metabolic activity with simultaneous depletion of TOC, phenolics and sugars. The main events of second phase are the increase of peroxidases production after the peak of laccase activity, and strong color removal. At the end of treatment, it was observed highly significant (p < 0.001) abatement of pollution parameters (83-100% removal). Since water reclamation and reuse for e.g. crop irrigation is a priority issue, vinasse ecotoxicity was assessed with bioindicators representing three different phylogenetic and trophic levels: a marine bacterium (Aliivibrio fischeri), a freshwater microcrustacean (Daphnia magna) and a dicotyledonous macrophyte (Lepidium sativum). It was observed significant (p < 0.05) reduction of initial vinasse toxicity, as evaluated by these bioindicators, deserving special mention an almost complete phytotoxicity elimination.

Growth conditions, physiological properties, and selection of optimal parameters of biodegradation of anticancer drug daunomycin in industrial effluents by Bjerkandera adusta CCBAS930.

The study characterizes the anamorphic Bjerkandera adusta strain CCBAS 930, including growth conditions, physiological properties, and enzymatic activities related to basic metabolism and specific properties coupled with the fungal secondary metabolism. It was established that the fungus grows in a wide pH range (3.5-7.5), up to 3% of salt concentration and a temperature of 5-30 °C. Media rich in natural organic components (potato, maize extracts, whey) are optimal for biomass propagation. Minimal media, containing mineral salts and glucose as well as static growth conditions, are required to obtain idiophasic mycelium, equivalent to the secondary metabolism of the fungus. Of the 7 complex C, N, and energy sources tested, the strain did not utilize only fibrous cellulose. Lipolytic activity reached the highest values of the enzymatic activities corresponding to those capabilities. The specific properties of strain B. adusta CCBAS 930 determined by the production of HRP-like peroxidase were related to the decolorization and biodegradation of anthraquinone derivative daunomycin. The decolorization of 30% of daunomycin effluents occurred most rapidly in iso-osmotic medium and non-enriched with nitrogen, containing 0.25% glucose, pH = 5.0-6.0, and 25-30 °C. In agitated cultures, the strain decolorized solutions of daunomycin by biosorption, which coincided with the inhibition of aerial mycelium production and HRP-like biosynthesis. Based on knowledge, potential and real possibilities of using the strain in bioremediation of colored industrial sewage were discussed.

Antitumour effect of glucooligosaccharides obtained via hydrolysis of α-(1 → 3)-glucan from Fomitopsis betulina.

Novel α-(1 → 3)-glucooligosaccharides (α-(1 → 3)-GOS) were prepared by acid hydrolysis of α-(1→ 3)-glucan isolated from Fomitopsis betulina fruiting bodies and characterized. Their anti-cancer potential was evaluated in in vitro assays in a colon cancer cell model. The tested α-(1 → 3)-GOS showed antiproliferative (MTT assay) and pro-apoptotic (Annexin V-FITC and PI technique) features against colon cancer but not against normal epithelial colon cells. Additionally, we did not observe cytotoxic activity (neutral red and lactate dehydrogenase assays) of α-(1 → 3)-GOS against several types of normal cell lines. In the present study, we demonstrated the anticancer potential of α-(1 → 3)-GOS in a colon carcinoma model. The anti-tumour effect of α-(1 → 3)-GOS is related with induction of apoptosis. Based on these results, we conclude that α-(1 → 3)-GOS may be considered as a dietary or therapeutic agent with an ability to inhibit the growth of cancer cells.

1H NMR-MS-based heterocovariance as a drug discovery tool for fishing bioactive compounds out of a complex mixture of structural analogues.

Chemometric methods and correlation of spectroscopic or spectrometric data with bioactivity results are known to improve dereplication in classical bio-guided isolation approaches. However, in drug discovery from natural sources the isolation of bioactive constituents from a crude extract containing close structural analogues remains a significant challenge. This study is a 1H NMR-MS workflow named ELINA (Eliciting Nature's Activities) which is based on statistical heterocovariance analysis (HetCA) of 1H NMR spectra detecting chemical features that are positively ("hot") or negatively ("cold") correlated with bioactivity prior to any isolation. ELINA is exemplified in the discovery of steroid sulfatase (STS) inhibiting lanostane triterpenes (LTTs) from a complex extract of the polypore fungus Fomitopsis pinicola.

Antimelanogenesis Effects of Fungal Exopolysaccharides Prepared from Submerged Culture of Fomitopsis castanea Mycelia.

Fungal exopolysaccharides are important natural products having diverse biological functions. In this study, exopolysaccharides from Fomitopsis castanea mycelia (FEPS) were prepared, and the highest mushroom tyrosinase inhibitory activity was found. FEPS were prepared from cultivation broth by ethanol precipitation method. The extraction yield and protein concentration of FEPS were 213.1 mg/l and 0.03%, respectively. FEPS inhibited mushroom tyrosinase with the half maximal inhibitory concentration (IC50) of 16.5 mg/ml and dose-dependently inhibited cellular tyrosinase activity (63.9% at 50 µg/ml, and 83.3% at 100 µg/ml) in the cell-free extract of SK-MEL-5 human melanoma cell and α-melanocytestimulating hormone (α-MSH)-stimulated melanin formation in intact SK-MEL-5 human melanoma cell. The IC50 of FEPS against NO production from RAW264.7 macrophage cells was 42.8 ± 0.64 µg/ml. By in vivo study using a zebrafish model, exposure of FEPS at 400 µg/ml to dechorionated zebrafish embryos for 18 h decreased the pigment density, compared to that without FEPS-treated control.

Modification of corn stover for improving biodegradability and anaerobic digestion performance by Ceriporiopsis subvermispora.

Ceriporiopsis subvermispora was used to modify corn stover for improving the biodegradability and biomethane yield. Corn stover was incubated with C. subvermispora for 5-90 days then anaerobically digested. It was found that the corn stover modified for 15 days achieved the highest biomethane yield of 235 mL·g-1 VS, which was an increase of 15.2% over that of the non-modified one. The mechanism analyses indicated that the improvement resulted from the combined roles of degradation selectivity, destruction of lignocellulosic structures, and linkages. The analyses showed that C. subvermispora has a high relative selectivity of lignin degradation. The structure of the lignin and the linkages among lignin and hemicellulose and cellulose were broken obviously by acetyl group removal, and the enzymatic hydrolysis of cellulose was increased by 35.61%. The finding indicated that C. subvermispora modification is one of the effective methods for enhancing biomethane yield of corn stover.

A GH family 28 endo-polygalacturonase from the brown-rot fungus Fomitopsis palustris: Purification, gene cloning, enzymatic characterization and effects of oxalate.

Brown-rot fungi are the wood-decay basidiomycetes and have ability to break down plant cell wall carbohydrates. It has been suggested that degradation of pectin is important for the initial stages of brown rot. We purified an endo-polygalacturonase (FpPG28A) from the brown-rot fungus Fomitopsis palustris, analysis of the predicted amino acid sequence indicated that FpPG28A belongs to GH family 28. The highest activity of purified FpPG28A was observed at 60 °C in 50 mM sodium acetate buffer (pH 5.0); this activity was highly specific for polygalacturonic acid chains. However, calcium polygalacturonate gel was not degraded by FpPG28A under those optimal conditions. We observed that calcium polygalacturonate gel was readily degraded by the enzyme in the oxalate buffer. Furthermore, the thermostability of FpPG28A was elevated in oxalate buffer at pH 3.0. These results indicated that oxalate has an important role in the degradation of woody pectin by FpPG28A.